Caenorhabditis elegans geminin homologue participates in cell cycle regulation and germ line development

Cdt1 is an essential component for the assembly of a pre-replicative complex. Cdt1 activity is inhibited by geminin, which also participates in neural development and embryonic differentiation in many eukaryotes. Although Cdt1 homologues have been identified in organisms ranging from yeast to human, geminin homologues had not been described for Caenorhabditis elegans and fungi. This study identifies the C. elegans geminin, GMN-1. Biochemical analysis reveals that GMN-1 associates with C. elegans CDT-1, the Hox protein NOB-1, and the Six protein CEH-32. GMN-1 inhibits not only the interaction between mouse Cdt1 and Mcm6 but also licensing activity in Xenopus egg extracts. RNA interference-mediated reduction of GMN-1 is associated with enlarged germ nuclei with aberrant nucleolar morphology, severely impaired gametogenesis, and chromosome bridging in intestinal cells. It is concluded that the Cdt1-geminin system is conserved throughout metazoans and that geminin has evolved in these taxa to regulate proliferation and differentiation by directly interacting with Cdt1 and homeobox proteins (Yanagi, 2005).

Geminin expression, inactivation and cell cycle

A novel 25 kDa protein, geminin, inhibits DNA replication and is degraded during the mitotic phase of the cell cycle. Geminin has a destruction box sequence and is ubiquitinated by anaphase-promoting complex (APC) in vitro. In synchronized HeLa cells, geminin is absent during G1 phase, accumulates during S, G2, and M phases, and disappears at the time of the metaphase-anaphase transition. Geminin inhibits DNA replication by preventing the incorporation of MCM complex into prereplication complex (pre-RC). It is proposed that geminin inhibits DNA replication during S, G2, and M phases and that geminin destruction at the metaphase-anaphase transition permits replication in the succeeding cell cycle (McGarry, 1998).

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase-anaphase transition and continues through G1 phase of the next cell cycle. Cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) have been used to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C-dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, injection of dominant negative Ubc3/Cdc34, whose role in G1-S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans (Bastians, 1999).

To ensure proper timing of the G1-S transition in the cell cycle, the cyclin E-Cdk2 complex, which is responsible for the initiation of DNA replication, is restrained by the p21(Cip1)/p27(Kip1)/p57(Kip2) family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27(Xic1) protein in Xenopus. Activation of cyclin E-Cdk2 is linked to the ubiquitination of human p27(Kip1) or Xenopus p27(Xic1) by SCF (for Skp1-Cullin-F-box protein) ubiquitin ligases. For human p27(Kip1), ubiquitination requires direct phosphorylation by cyclin E-Cdk2. Xic1 ubiquitination does not require phosphorylation by cyclin E-Cdk2, but it does require nuclear accumulation of the Xic1-cyclin E-Cdk2 complex and recruitment of this complex to chromatin by the origin-recognition complex together with Cdc6 replication preinitiation factors; it also requires an activation step necessitating cyclin E-Cdk2-kinase and SCF ubiquitin-ligase activity, and additional factors associated with mini-chromosome maintenance proteins, including the inactivation of geminin. Components of the SCF ubiquitin-ligase complex, including Skp1 and Cul1, are also recruited to chromatin through cyclin E-Cdk2 and the preinitiation complex. Thus, activation of the cyclin E-Cdk2 kinase and ubiquitin-dependent destruction of its inhibitor are spatially constrained to the site of a properly assembled preinitiation complex (Furstenthal, 2001).

The behavior of pre-replication complex (pre-RC) proteins have been examined in relation to key cell cycle transitions in Chinese hamster ovary (CHO) cells. ORC1, ORC4 and Cdc6 are stable (T1/2 >2 h) and associate with a chromatin-containing fraction throughout the cell cycle. Green fluorescent protein-tagged ORC1 associates with chromatin throughout mitosis in living cells and co-localizes with ORC4 in metaphase spreads. Association of Mcm proteins with chromatin takes place during telophase, approximately 30 min after the destruction of geminin and cyclins A and B, and is coincident with the licensing of chromatin to replicate in geminin-supplemented Xenopus egg extracts. Neither Mcm recruitment nor licensing requires protein synthesis throughout mitosis. Moreover, licensing can be uncoupled from origin specification in geminin-supplemented extracts; site-specific initiation within the dihydrofolate reductase locus requires nuclei from cells that have passed through the origin decision point (ODP). These results demonstrate that mammalian pre-RC assembly takes place during telophase, mediated by post-translational modifications of pre-existing proteins, and is not sufficient to select specific origin sites. A subsequent, as yet undefined, step selects which pre-RCs will function as replication origins (Okuno, 2001).

A hypomorphic mutation made in the ORC2 gene of a human cancer cell line through homologous recombination decreases Orc2 protein levels by 90%. The G1 phase of the cell cycle is prolonged, but there is no effect on the utilization of either the c-Myc or beta-globin cellular origins of replication. Cells carrying this mutation fail to support the replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV), and this defect is rescued by reintroduction of Orc2. Orc2 specifically associates with oriP in cells, most likely through its interaction with EBNA1. Geminin, an inhibitor of the mammalian replication initiation complex, inhibits replication from oriP. Therefore, ORC and the human replication initiation apparatus is required for replication from a viral origin of replication (Dhar, 2001).

Association of Geminin with Cdt1

In all eukaryotic organisms, inappropriate firing of replication origins during the G2 phase of the cell cycle is suppressed by cyclin-dependent kinases. Multicellular eukaryotes contain a second putative inhibitor of re-replication, called geminin. Geminin is believed to block binding of the mini-chromosome maintenance (MCM) complex to origins of replication, but the mechanism of this inhibition is unclear. Geminin is shown to interact tightly with Cdt1 (See Drosophila Double parked), a recently identified replication initiation factor necessary for MCM loading. The inhibition of DNA replication by geminin that is observed in cell-free DNA replication extracts is reversed by the addition of excess Cdt1. In the normal cell cycle, Cdt1 is present only in G1 and S, whereas geminin is present in S and G2 phases of the cell cycle. Together, these results suggest that geminin inhibits inappropriate origin firing by targeting Cdt1 (Wohlschlegel, 2000).

Eukaryotic replication origins are 'licensed' for replication early in the cell cycle by loading Mcm(2-7) proteins. As chromatin replicates, Mcm(2-7) proteins are removed, thus preventing the origin from firing again. The purification of the RLF-B component of the licensing system has been purified; it corresponds to Cdt1. RLF-B/Cdt1 is inhibited by geminin, a protein that is degraded during late mitosis. Immunodepletion of geminin from metaphase extracts allows them to assemble licensed replication origins. Inhibition of CDKs in metaphase stimulates origin assembly only after the depletion of geminin. These experiments suggest that geminin-mediated inhibition of RLF-B/Cdt1 is essential for repressing origin assembly late in the cell cycle of higher eukaryotes (Tada, 2001).

S-phase onset is controlled, so that it occurs only once every cell cycle. DNA is licensed for replication after mitosis in G1, and passage through S-phase removes the license to replicate. In fission yeast, Cdc6/18 and Cdt1, two factors required for licensing, are central to ensuring that replication occurs once per cell cycle. The human Cdt1 homolog (hCdt1), a nuclear protein, is present only during G1. After S-phase onset, hCdt1 levels decrease, and it is hardly detected in cells in early S-phase or G2. hCdt1 can associate with the DNA replication inhibitor Geminin, however these two proteins are mostly expressed at different cell cycle stages. hCdt1 mRNA, in contrast to hCdt1 protein, is expressed in S-phase-arrested cells, and its levels do not change dramatically during a cell cycle, suggesting that proteolytic rather than transcriptional controls ensure the timely accumulation of hCdt1. Consistent with this view, proteasome inhibitors stabilize hCdt1 in S-phase. In contrast, hCdc6/18 levels are constant through most of the cell cycle and are only low for a brief period at the end of mitosis. These results suggest that the presence of active hCdt1 may be crucial for determining when licensing is legitimate in human cells (Nishitani, 2001).

During late mitosis and early interphase, origins of replication become 'licensed' for DNA replication by loading Mcm2-7 complexes. Mcm2-7 complexes are removed from origins as replication forks initiate replication, thus preventing rereplication of DNA in a single cell cycle. Premature origin licensing is prevented in metaphase by the action of geminin, which binds and inhibits Cdt1/RLF-B, a protein that is required for the loading of Mcm2-7. Recombinant geminin that is added to Xenopus egg extracts is efficiently degraded upon exit from metaphase. Recombinant and endogenous forms of Xenopus geminin behave differently from one another, such that a significant proportion of endogenous geminin escapes proteolysis upon exit from metaphase. During late mitosis and early G1, the surviving population of endogenous geminin does not associate with Cdt1/RLF-B and does not inhibit licensing. Following nuclear assembly, geminin is imported into nuclei and becomes reactivated to bind Cdt1/RLF-B. This reactivated geminin provides the major nucleoplasmic inhibitor of origin relicensing during late interphase. Thus, upon metaphase release, some of the geminin is degraded, but the remaining geminin is altered in some way that prevents it from binding and inhibiting Cdt1/RLF-B. The loss of CDK activity allows ORC and Cdc6 to associate tightly with DNA. With all components of the licensing system active, Mcm2-7 is loaded onto origins. Following nuclear assembly, geminin is imported into a functional nucleus and becomes reactivated. This allows it to bind and inhibit Cdt1/RLF-B, thus preventing the relicensing of replicated origins. Since the initiation of replication at licensed origins depends on nuclear assembly, these results suggest an elegant and novel mechanism for preventing rereplication of DNA in a single cell cycle (Hodgson, 2002).

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. How rereplication is prevented in normal mammalian cells has been investigated; these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 (Drosophila homolog: Double parked) and Cdc6 (Drosophila homolog: Origin recognition complex subunit 1) along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability (Vaziri, 2003).

To test whether geminin inhibits rereplication induced by Cdt1, geminin was overexpressed along with Cdt1 and Cdc6. Overexpression of geminin partially inhibits the rereplication mediated by Cdt1+Cdc6. Overexpression of Cdt1 (by itself) leads to a paradoxical increase in geminin levels in the rereplicating cells. In order to confirm that there was free Cdt1 (uncomplexed with geminin) in the cell lines, all the geminin was precleared from these cell extracts before immunoblotting for residual Cdt1 in the supernatant. The results show that despite the induction of geminin, not enough of the protein is produced to associate with and inhibit all the overexpressed Cdt1. The increase in geminin was attributed to a 10-fold induction of geminin mRNA seen upon overexpression of Cdt1. The mechanism of this induction is currently unclear but suggests the existence of a feedback loop between Cdt1 and its antagonist geminin (Vaziri, 2003).

The activation of the DNA damage checkpoint pathway and the tumor suppressor protein p53 provides a pathway by which mammalian cells prevent rereplication. Rereplication appears to lead to DNA damage. The data suggest that activation of ATM/ATR kinases caused by overexpression of Cdt1 and Cdc6 leads to direct phosphorylation of p53 and indirect phosphorylation of p53 through Chk2 kinase. Phosphorylation of p53 stabilizes the protein and leads to increased transcription and expression of p21. The latter is a potent inhibitor of cyclin A-cdk2 kinase and could therefore prevent any rereplication. Consistent with this hypothesis, overexpression of wild-type p53 or of p21 effectively inhibits rereplication in the p53-negative H1299 cells, while inactivation of p53 in A549 cells by overexpressing Mdm2 prevents p21 induction and permits rereplication. Because of the concurrent induction of proapoptotic genes like PIG3, p53 could also promote apoptosis of cells that have already undergone significant rereplication. Since mutations in p53 have been widely documented to promote genomic instability and gene amplification, these results provide a partial explanation of this observation by proposing a mechanism by which p53 stabilizes the genome. Genes other than p53, however, also prevent gene amplification, so it is unlikely that p53 is the only barrier to rereplication upon overexpression of Cdt1 and Cdc6 in all cell lines (Vaziri, 2003).

Geminin is an unstable inhibitor of DNA replication that negatively regulates the licensing factor CDT1 and inhibits pre-replicative complex (pre-RC) formation in Xenopus egg extracts. A novel function of Geminin is described. Human Geminin protects CDT1 from proteasome-mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle. Moreover, inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. These results demonstrate that Geminin is both a negative and positive regulator of pre-RC formation in human cells, playing a positive role in allowing CDT1 accumulation in G2-M, and preventing relicensing of origins in S-G2 (Ballabeni, 2004).

Geminin is an unstable regulatory protein that affects both cell division and cell differentiation. Geminin inhibits a second round of DNA synthesis during S and G(2) phase by binding the essential replication protein Cdt1. Geminin is also required for entry into mitosis, either by preventing replication abnormalities or by down-regulating the checkpoint kinase Chk1. Geminin overexpression during embryonic development induces ectopic neural tissue, inhibits eye formation, and perturbs the segmental patterning of the embryo. In order to define the structural and functional domains of the geminin protein, over 40 missense and deletion mutations were generated and their phenotypes were tested in biological and biochemical assays. Teminin self-associates through the coiled-coil domain to form dimers and dimerization is required for activity. Geminin contains a typical bipartite nuclear localization signal that is also required for its destruction during mitosis. Nondegradable mutants of geminin interfere with DNA replication in succeeding cell cycles. Geminin's Cdt1-binding domain lies immediately adjacent to the dimerization domain and overlaps it. Two nonbinding mutants in this domain were constructed and they were found to neither inhibit replication nor permit entry into mitosis, indicating that this domain is necessary for both activities. Several missense mutations in geminin's Cdt1 binding domain were identified that were deficient in their ability to inhibit replication yet were still able to allow mitotic entry, suggesting that these are separate functions of geminin (Benjamin, 2004).

To maintain chromosome stability in eukaryotic cells, replication origins must be licensed by loading mini-chromosome maintenance (MCM2-7) complexes once and only once per cell cycle. This licensing control is achieved through the activities of geminin and cyclin-dependent kinases. Geminin binds tightly to Cdt1, an essential component of the replication licensing system, and prevents the inappropriate reinitiation of replication on an already fired origin. The inhibitory effect of geminin is thought to prevent the interaction between Cdt1 and the MCM helicase. The crystal structure of the mouse geminin-Cdt1 complex is described using tGeminin (residues 79-157, truncated geminin) and tCdt1 (residues 172-368, truncated Cdt1). The amino-terminal region of a coiled-coil dimer of tGeminin interacts with both N-terminal and carboxy-terminal parts of tCdt1. The primary interface relies on the steric complementarity between the tGeminin dimer and the hydrophobic face of the two short N-terminal helices of tCdt1 and, in particular, Pro 181, Ala 182, Tyr 183, Phe 186 and Leu 189. The crystal structure, in conjunction with biochemical data, indicates that the N-terminal region of tGeminin might be required to anchor tCdt1, and the C-terminal region of tGeminin prevents access of the MCM complex to tCdt1 through steric hindrance (Lee, 2004).

To maintain chromosome stability in eukaryotic cells, replication origins must be licensed by loading mini-chromosome maintenance (MCM2-7) complexes once and only once per cell cycle. This licensing control is achieved through the activities of geminin and cyclin-dependent kinases. Geminin binds tightly to Cdt1, an essential component of the replication licensing system, and prevents the inappropriate reinitiation of replication on an already fired origin. The inhibitory effect of geminin is thought to prevent the interaction between Cdt1 and the MCM helicase. The crystal structure of the mouse geminin-Cdt1 complex is described using tGeminin (residues 79-157, truncated geminin) and tCdt1 (residues 172-368, truncated Cdt1). The amino-terminal region of a coiled-coil dimer of tGeminin interacts with both N-terminal and carboxy-terminal parts of tCdt1. The primary interface relies on the steric complementarity between the tGeminin dimer and the hydrophobic face of the two short N-terminal helices of tCdt1 and, in particular, Pro 181, Ala 182, Tyr 183, Phe 186 and Leu 189. The crystal structure, in conjunction with biochemical data, indicates that the N-terminal region of tGeminin might be required to anchor tCdt1, and the C-terminal region of tGeminin prevents access of the MCM complex to tCdt1 through steric hindrance (Lee, 2004).

Geminin is a cellular protein that associates with Cdt1 and inhibits Mcm2-7 loading during S phase. It prevents multiple cycles of replication per cell cycle and prevents episome replication. It also directly inhibits the HoxA11 transcription factor. Geminin forms a parallel coiled-coil homodimer with atypical residues in the dimer interface. Point mutations that disrupt the dimerization abolish interaction with Cdt1 and inhibition of replication. An array of glutamic acid residues on the coiled-coil domain surface interacts with positive charges in the middle of Cdt1. An adjoining region interacts independently with the N-terminal 100 residues of Cdt1. Both interactions are essential for replication inhibition. The negative residues on the coiled-coil domain and a different part of geminin are also required for interaction with HoxA11. Therefore a rigid cylinder with negative surface charges is a critical component of a bipartite interaction interface between geminin and its cellular targets (Saxena, 2004).

In late mitosis and G1, Mcm2-7 are assembled onto replication origins to 'license' them for initiation. At other cell cycle stages, licensing is inhibited, thus ensuring that origins fire only once per cell cycle. Three additional factors -- the origin recognition complex, Cdc6 and Cdt1 -- are required for origin licensing. This study examines how licensing is regulated in Xenopus egg extracts. Cdt1 is shown to be downregulated late in the cell cycle by two different mechanisms: proteolysis, which occurs in part due to the activity of the anaphase-promoting complex (APC/C), and inhibition by geminin. If both these regulatory mechanisms are abrogated, extracts undergo uncontrolled re-licensing and re-replication. The extent of re-replication is limited by checkpoint kinases that are activated as a consequence of re-replication itself. These results allow the building of a comprehensive model of how re-replication of DNA is prevented in Xenopus, with Cdt1 regulation being the key feature. The results also explain the original experiments that led to the proposal of a replication licensing factor (Li, 2005).

Cdt1 plays a key role in licensing DNA for replication. In the somatic cells of metazoans, both Cdt1 and its natural inhibitor geminin show reciprocal fluctuations in their protein levels owing to cell cycle-dependent proteolysis. This study shows that the protein levels of Cdt1 and geminin are persistently high during the rapid cell cycles of the early Xenopus embryo. Immunoprecipitation of Cdt1 and geminin complexes, together with their cell cycle spatiotemporal dynamics, strongly supports the hypothesis that Cdt1 licensing activity is regulated by periodic interaction with geminin rather than its proteolysis. Overexpression of ectopic geminin slows down, but neither arrests early embryonic cell cycles nor affects endogenous geminin levels; apparent embryonic lethality is observed around 3-4 hours after mid-blastula transition. However, functional knockdown of geminin by δCdt1_193-447, which lacks licensing activity and degradation sequences, causes cell cycle arrest and DNA damage in affected cells. This contributes to subsequent developmental defects in treated embryos. The results clearly show that rapidly proliferating early Xenopus embryonic cells are able to regulate replication licensing in the persistent presence of high levels of licensing proteins by relying on changing interactions between Cdt1 and geminin during the cell cycle, but not their degradation (Kisielewska, 2011).

The APC/C inhibitor, Emi1, is essential for prevention of rereplication

Emi1 (early mitotic inhibitor) inhibits APC/C (anaphase-promoting complex/cyclosome) activity during S and G2 phases, and is believed to be required for proper mitotic entry. Emi1 plays an essential function in cell proliferation by preventing rereplication. Rereplication seen after Emi1 depletion is due to premature activation of APC/C that results in destabilization of geminin and cyclin A, two proteins shown in this study to play redundant roles in preventing rereplication in mammalian cells. Geminin is known to inhibit the replication initiation factor Cdt1. The rereplication block by cyclin A is mediated through its association with S and G2/M cyclin-dependent kinases (Cdks), Cdk2 and Cdk1, suggesting that phosphorylation of proteins by cyclin A-Cdk is responsible for the block. Rereplication upon Emi1 depletion activates the DNA damage checkpoint pathways. These data suggest that Emi1 plays a critical role in preserving genome integrity by blocking rereplication, revealing a previously unrecognized function of this inhibitor of APC/C (Machida, 2007).

DNA replication initiates from the specific regions of chromosomal DNA called origins. A critical question in the field of genomic stability is how cells manage to restrict the firing of origins to once and only once per cell cycle. Not only is this regulation achieved despite the fact that thousands of origins fire asynchronously all over the genome, but the regulation is selectively breached during certain stages of development as in endoreduplicating trophoblasts. Most of this regulation appears to be executed at the level of prereplicative complex (pre-RC) formation or origin licensing. The origin recognition complex (ORC) recruits Cdc6 and Cdt1 to origins in G1, which in turn load the putative replicative helicase, Mcm2-7, to form pre-RCs. After replication initiation in the subsequent S phase, Mcm2-7 are depleted from origins, but reformation of pre-RCs is prevented until chromosomes are segregated in M phase. Higher eukaryotes, including human cells, express geminin a protein that binds to Cdt1 to prevent loading of Mcm2-7 on post-initiation origins. Knockdown of geminin by RNA interference (RNAi) is sufficient to induce rereplication in many human cell lines, and geminin-null mice show enhanced endoreduplication in trophoblasts of early embryos. These results suggest that geminin is a major inhibitor of rereplication in mammalian cells. Yeast, however, do not encode geminin, and rereplication is prevented solely by the high levels cyclin-dependent kinase (Cdk) activity seen during S and G2 phases of the cell cycle. A role of high Cdk activity in rereplication block has also been suggested in human cells, although cancer cells that show rereplication after geminin knockdown do so without any obvious mechanism to concurrently inhibit Cdk2 activity. It is therefore currently unclear whether geminin and Cdk play redundant roles in rereplication block, or whether these mechanisms function in different parts of the cell cycle or in different tissues (Machida, 2007 and references therein).

The regulated degradation of proteins by proteasomes through a carefully orchestrated polyubiquitination program is a critical component of cell cycle regulation. For example, Cdks are important for progression through S, G2, and M, but their activity is regulated by the periodic accumulation and destruction of different types of cyclins. Levels of cyclins in the cell cycle are regulated by two ubiquitin ligases, SCF and anaphase-promoting complex/cyclosome (APC/C). SCF complex uses an F-box protein as a substrate recognition subunit. For example, SCFFBXW7 polyubiquitinates the G1 cyclin, cyclin E, for degradation in S phase. There are >60 F-box proteins in the human genome but the cellular functions of most of them are not yet known. APC/C, on the other hand, uses substrate recognition subunits Cdc20 and Cdh1 to polyubiquitinate substrates like cyclins A and B (and geminin) in mitosis and the subsequent G1. Cdc20 activates APC/C in mitosis while Cdh1 activates APC/C in late M and G1 phases. Both these subunits target substrates by recognizing a destruction motif called D-box, while Cdh1, in addition, recognizes a KEN-box (Machida, 2007 and references therein).

Since cyclin A/Cdk kinase activity is essential for DNA replication, the inactivation of APC/C at the G1/S transition is critical for the accumulation of cyclin A for S-phase progression. Emi1 (early mitotic inhibitor) is a cellular inhibitor of APC/C that is not present in yeasts, and is induced by the E2F transcription factor to inactivate APC/C at the G1/S transition. Emi1-mediated reduction in APC/C activity allows cells to accumulate cyclin A, and then the accumulated cyclin A-Cdk complex in S phase further suppresses APC/C activity by phosphorylating Cdh1. Because of its role in suppression of APC/C and because the latter is critical for mitosis, the primary role of Emi1 is believed to ensure proper mitotic entry. At the onset of mitosis, Emi1 is degraded following phosphorylation by Plk1 and ubiquitination by SCF-TrCP, and this allows the activation of APC/C that is critical for progression through M phase and the subsequent G1 phase (Machida, 2007 and references therein).

Since SCF is involved in many aspects of cell cycle regulation, a search was initiated for F-box proteins that are required for cell proliferation by RNAi screening. This screen identified Emi1, an F-box protein known as FBXO5, to be essential for cell proliferation. Surprisingly, the block to cell proliferation was accompanied by extensive rereplication after Emi1 depletion. This rereplication is due to premature activation of APC/C in S and G2 cells. Among many APC/C substrates, cyclin A and geminin are critical for prevention of rereplication, and the two proteins act simultaneously in S and G2 cells as redundant barriers to rereplication. These data suggest that an essential function of Emi1 in S and G2 cells is to prevent rereplication via stabilization of inhibitors of rereplication such as cyclin A and geminin (Machida, 2007).

Transcription regulation of geminin

Geminin and Cdt1 play an essential role in the initiation of DNA replication, by regulating the chromatin loading of the MCM complex. The transcription of human Geminin and Cdt1, as well as that of MCM7, is activated by transcription factors E2F1-4, but not by factors E2F5-7. Analysis of various Geminin and Cdt1 promoter constructs shows that an E2F-responsive sequence in the vicinity of the transcription initiation site is necessary for the transcriptional activation. The promoter activity for human Geminin was activated by the E7, but not E6, oncogene of human papillomavirus type 16. While E2F1-induced activation of human Cdt1 gene transcription was suppressed by pRb, but not by p107 or p130, its E2F4-induced activation was suppressed by pRb, p107, and p130. Furthermore, the promoter activities of human Geminin and Cdt1 were demonstrated to be growth-dependent. Taken together, the results demonstrate that Geminin and Cdt1 constitute targets for various members of the E2F family of transcription factors, and that expression of Geminin and Cdt1 is perhaps mediated by the activation of a pRb/E2F pathway (Yoshida, 2004).

Geminin inhibits Hox function through direct and polycomb-mediated interactions

Embryonic development is tightly controlled. The clustered genes of the Hox family of homeobox proteins play an important part in regulating this development and also proliferation. They specify embryonic structures along the body axis, and are associated with normal and malignant cell growth. The cell-cycle regulator geminin controls replication by binding to the licensing factor Cdt1, and is involved in neural differentiation. Murine geminin associates transiently with members of the Hox-repressing polycomb complex, with the chromatin of Hox regulatory DNA elements and with Hox proteins. Gain- and loss-of-function experiments in the chick neural tube demonstrate that geminin modulates the anterior boundary of Hoxb9 transcription, which suggests a polycomb-like activity for geminin. The interaction between geminin and Hox proteins prevents Hox proteins from binding to DNA, inhibits Hox-dependent transcriptional activation of reporter and endogenous downstream target genes, and displaces Cdt1 from its complex with geminin. By establishing competitive regulation, geminin functions as a coordinator of developmental and proliferative control (Luo, 2004).

Geminin, development and differentation

In an expression cloning screen in Xenopus embryos, a gene has been identified that when overexpressed expands the neural plate at the expense of adjacent neural crest and epidermis. This gene, termed geminin, had no sequence similarity to known gene families. Geminin's neuralizing domain is part of a bifunctional protein whose C-terminal coiled-coil domain may play a role in regulating DNA replication. The neuralizing function of geminin is described in this study. The localization, effect of misexpression and activity of a dominant negative geminin suggest that the product of this gene has an essential early role in specifying neural cell fate in vertebrates. Maternal geminin mRNA is found throughout the animal hemisphere from oocyte through late blastula. At the early gastrula, however, expression is restricted to a dorsal ectodermal territory that prefigures the neural plate. Misexpression of geminin in gastrula ectoderm suppresses BMP4 expression and converts prospective epidermis into neural tissue. In ectodermal explants, geminin induces expression of the early proneural gene neurogenin-related 1 although not itself being induced by that gene. Later, embryos expressing geminin have an expanded dorsal neural territory and ventral ectoderm is converted to neurons. A putative dominant negative geminin lacking the neuralizing domain suppresses neural differentiation and, when misexpressed dorsally, produces islands of epidermal gene expression within the neurectodermal territory, effects that are rescued by coexpression of the full-length molecule. Taken together, these data indicate that geminin plays an early role in establishing a neural domain during gastrulation (Kroll, 1998).

Two functional domains of geminin were defined in embryos by testing a series of geminin deletion mutants for activity. An N-terminal domain (amino acids 38-90; Ngem) was sufficient to evoke neural hypertrophy and ectopic neurogenesis when injected at RNA levels slightly higher than those required for the full-length molecule. A C-terminal domain comprised of the coiled coils of the molecule (amino acids 112-168; Ccoil) was cytotoxic when expressed in early embryos. This domain has DNA replication inhibition activity and can fully account for geminin's ability to regulate DNA replication in vitro. By contrast, the N- terminal domain has no effect on DNA replication or cell cycle progression, is not cytotoxic in embryos at doses up to 2 ng and can fully account for geminin’s neuralizing activity. These cDNAs also contain a destruction box that is near but not contained in the N-terminal neuralizing fragment. The neuralizing and cell cycle regulatory activities of geminin are physically separated into non-overlapping and independently acting domains. However, alteration of the cell cycle state could conceivably be a precondition for neural determination or differentiation such that physical association of these domains would be advantageous. To test whether blocking the cell cycle elicites ectopic neurogenesis, embryos were treated with hydroxyurea and aphidicolin (HUA) from the early gastrula stage. This HUA treatment blocks virtually all cell division and does not elicit the neural hypertrophy found in previous studies. HUA treatment alone has no effect on neural plate formation or expression of neural genes. Neural hypertrophy elicited by geminin is not attributable to increased cell division as it occurs in HUA-treated embryos previously injected with geminin mRNA. Therefore, the neuralizing effects of geminin are neither sensitive to nor caused by perturbation of the cell cycle (Kroll, 1998).

Homologs of the murine Brachyury gene have been shown to be involved in mesoderm formation in several vertebrate species. In frogs, the Xenopus Brachyury homolog, Xbra, is required for normal formation of posterior mesoderm. A second Brachyury homolog from Xenopus, Xbra3, has been identified, which has levels of identity with mouse Brachyury similar to those of Xbra. Xbra3 encodes a nuclear protein expressed in mesoderm in a temporal and spatial manner distinct from that observed for Xbra. Xbra3 expression is induced by mesoderm-inducing factors and overexpression of Xbra3 can induce mesoderm formation in animal caps. In contrast to Xbra, Xbra3 is also able to cause the formation of neural tissue in animal caps. Xbra3 overexpression induces both geminin and Xngnr-1, suggesting that Xbra3 can play a role in the earliest stages of neural induction. Xbra3 induces posterior nervous tissue by an FGF-dependent pathway; a complete switch to anterior neural tissue can be effected by the inhibition of FGF signaling. Neither noggin, chordin, follistatin, nor Xnr3 is induced by Xbra3 to an extent different from their induction by Xbra nor is BMP4 expression differentially affected (Strong, 2001).

Organogenesis in vertebrates requires the tight control of cell proliferation and differentiation. The homeobox-containing transcription factor Six3 plays a pivotal role in the proliferation of retinal precursor cells. In a yeast two-hybrid screen, the DNA replication-inhibitor geminin has been identified as a partner of Six3. Geminin inhibits cell-cycle progression by sequestering Cdt1, the key component for the assembly of the pre-replication complex. Six3 efficiently competes with Cdt1 directly to bind to geminin, which reveals how Six3 can promote cell proliferation without transcription. In common with Six3 inactivation, overexpression of the geminin gene (Gem; also known as Gmn) in medaka (Oryzias latipes) induces specific forebrain and eye defects that are rescued by Six3. Conversely, loss of Gem (in common with gain of Six3) promotes retinal precursor-cell proliferation and results in expanded optic vesicles, markedly potentiating Six3 gain-of-function phenotypes. These data indicate that the transcription factor Six3 and the replication-initiation inhibitor geminin act antagonistically to control the balance between proliferation and differentiation during early vertebrate eye development (Del Bene, 2004).

Precise control of cell proliferation and differentiation is critical for organogenesis. Geminin (Gem) has been proposed to link cell cycle exit and differentiation as a prodifferentiation factor and plays a role in neural cell fate acquisition. The SWI/SNF chromatin-remodeling protein Brg1 has been identified as an interacting partner of Gem. Brg1 has been implicated in cell cycle withdrawal and cellular differentiation. Surprisingly, Gem was found to antagonize Brg1 activity during neurogenesis to maintain the undifferentiated cell state. Down-regulation of Gem expression normally precedes neuronal differentiation, and gain- and loss-of-function experiments in Xenopus embryos and mouse P19 cells demonstrate that Gem is essential to prevent premature neurogenesis. Misexpression of Gem also suppresses ectopic neurogenesis driven by Ngn and NeuroD. Gem's activity to block differentiation depends upon its ability to bind Brg1 and could be mediated by Gem's inhibition of proneural basic helix-loop-helix (bHLH) Brg1 interactions required for bHLH target gene activation. The data demonstrate a novel mechanism of Gem activity, through regulation of SWI/SNF chromatin-remodeling proteins, and indicate that Gem is an essential regulator of neurogenesis that can control the timing of neural progenitor differentiation and maintain the undifferentiated cell state (Seo, 2005).

Ngn and NeuroD proteins interact directly with Brg1 and require Brg1 activity to activate target gene transcription. Since Gem interacts with Brg1, whether Gem could form a higher-order complex with Brg1 and bHLH proteins or could compete with bHLH proteins for Brg1 binding was examined. In transfection and co-IP experiments, no bHLH-Gem interactions were found, while association of Brg1 and Ngn/NeuroD was observed. Therefore, it is unlikely that Gem forms a complex together with bHLH factors and Brg1. Instead, overexpression of wild-type Gem can inhibit the association of Ngn and NeuroD with Brg1. The ability of Gem to block Ngn/NeuroD and Brg1 interaction is strongly reduced for GemDelta(BD), indicating that this activity requires an intact Brg1-binding motif. In addition, while wild-type Gem can suppress the ability of Ngn3 to activate target gene transcription, GemDelta(BD) cannot. These data suggest that Gem can suppress neuronal differentiation, at least partly, by blocking association of proneural bHLHs and Brg1, and thus preventing transcriptional activation of target genes (Seo, 2005).

Geminin, cell cycle and cancer

Licensing origins for replication upon completion of mitosis ensures genomic stability in cycling cells. Cdt1 was recently discovered as an essential licensing factor, which is inhibited by geminin. Over-expression of Cdt1 predisposes cells for malignant transformation. Cdt1 is down-regulated at both the protein and RNA level when primary human fibroblasts exit the cell cycle into G0, and its expression is induced as cells re-enter the cell cycle, prior to S phase onset. Cdt1's inhibitor, geminin, is similarly down-regulated upon cell cycle exit at both the protein and RNA level, and geminin protein accumulates with a 3-6 h delay over Cdt1, following serum re-addition. Similarly, mouse NIH3T3 cells down-regulate Cdt1 and geminin mRNA and protein when serum starved. The data suggest a transcriptional control over Cdt1 and geminin at the transition from quiescence to proliferation. In situ hybridization and immunohistochemistry localize Cdt1 as well as geminin to the proliferative compartment of the developing mouse gut epithelium. Cdt1 and geminin levels were compared in primary cells vs. cancer-derived human cell lines. Cdt1 is consistently over-expressed in cancer cell lines at both the protein and RNA level, and the Cdt1 protein accumulates to higher levels in individual cancer cells. Geminin is similarly over-expressed in the majority of cancer cell lines tested. The relative ratios of Cdt1 and geminin differ significantly amongst cell lines. These data establish that Cdt1 and geminin are regulated at cell cycle exit, and suggest that the mechanisms controlling Cdt1 and geminin levels may be altered in cancer cells (Xouri, 2004).

geminin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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